Effect and Mechanism of Anlotinib on the Malignant Biological Behavior of Cisplatin-resistant Cells in Cervical Cancer

doi:10.21203/rs.3.rs-3784746/v1

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Effect and Mechanism of Anlotinib on the Malignant Biological Behavior of Cisplatin-resistant Cells in Cervical Cancer

https://doi.org/10.21203/rs.3.rs-3784746/v1

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Purpose

To explore the effect of anlotinib on the proliferation, apoptosis, migration and invasion of cisplatin-resistant cervical cancer cells and study the changes of gene transcriptome of cisplatin-resistant cervical cancer cell lines after intervention with anlotinib.

Materials and Methods

In this study, cisplatin was used to induce the establishment of a drug-resistant cervical cancer cell line Siha/DDP. Then we use CCK-8 test, cell cloning test, flow cytometry test and western blotting test to explore the effect of anlotinib on the proliferation and apoptosis of Siha/DDP cells. Cell scratch test and transwell test were used to explore the effect of anlotinib on the migration and invasion of Siha/DDP cells. Finally transcriptome sequencing was used to detect changes in the gene expression profile of Siha/DDP cells under the action of anlotinib.

Results

Compared with the control group, the anlotinib group was able to dose-dependently inhibit Siha/DDP cell proliferation, migration, and invasion and significantly inhibit Siha/DDP cell clone formation; it was also able to dose-dependently promote Siha/DDP cell apoptosis and block Siha/DDP cells in the G2/M phase. Anlotinib significantly upregulated Bax, P21, E-cadherin protein expression and significantly downregulated Bcl-2, CDK1 and CyclinB1, VEGF, MMP-9 protein and drug resistance-associated protein P-gp protein expression in Siha/DDP cells. The gene expression profile of Siha/DDP cells caused changes after treatment with anlotinib. SQLE, MSMO1, LSS, FDFT1 and HMGCS1 were obtained as key hub gene

Conclusion

Anlotinib can inhibit the proliferation, migration and invasion of Siha/DDP cells and promote their apoptosis. The gene expression profile of Siha/DDP cells was altered after treatment with anlotinib, causing differential expression of several genes including SQLE, MSMO1, LSS, FDFT1 and HMGCS1.

cervical cancer

anlotinib

cisplatin

drug resistance

mechanism of action

Cervical cancer is the most common gynecological malignancy with a high incidence rate, which seriously affects women's life expectancy and quality of life. According to WHO data, there will be 604,000 new cases of cervical cancer worldwide in 2020, and 109,000 new cases in China; there will be 342,000 cases of cervical cancer deaths worldwide, accounting for 4.7% of female tumor deaths, of which the number of deaths in China is 59,000(Sung et al. 2021, Li et al. 2020). Clinically, according to the clinical stage and systemic condition of cervical cancer patients, surgery is the main treatment for patients with IA ~ IIA, while radiotherapy is the main treatment for patients with advanced stage IIB and III, stage IV or inoperable, but chemotherapy is mostly used for patients with late stage or recurrent metastasis and simultaneous radiotherapy and chemotherapy(Haritha et al. 2022).

Cisplatin is commonly used in chemotherapy for cervical cancer(Kumar et al. 2018), however, the effect of combination therapy for cervical cancer is still limited due to radiotherapy resistance and chemoresistance(Zhu et al. 2016), and how to improve the sensitivity of cervical cancer cells to cisplatin drugs is a challenge that plagues clinical treatment.

Anlotinib is a novel oral small-molecule multi-target tyrosine kinase inhibitor developed independently in China(Guanghua and Yu 2022), which has a broad inhibitory effect on tumor angiogenesis and tumor cell growth. Anlotinib has not been approved as an indication for the treatment of gynecologic tumors, but several clinical studies have shown that anlotinib has good efficacy and safety in the treatment of gynecologic tumors. A study first evaluated the efficacy of anlotinib in Chinese patients with recurrent or metastatic CC and found durable efficacy and a controlled safety profile (Zhu et al. 2021). A retrospective study data showed that anlotinib was safe and effective in patients with advanced cervical cancer after radiotherapy and was well tolerated(Tan et al. 2021).

The aim of this study was to investigate the mechanism of action of anlotinib alone and its combination with cisplatin in drug-resistant strains of cervical cancer, with the aim of providing new ideas for targeted therapy in patients with advanced and recurrent cervical cancer.

Cell line

Human cervical cancer Siha cell line was obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China).

The main reagents

Anlotinib was purchased from MedChemExpress (USA). Cisplatin (DDP) was purchased from Shandong Qilu Pharmaceutical Co. Dimethyl sulfoxide (DMSO) was purchased from Sigma (U.S.A). CCK-8 reagent was purchased from MedChemExpress (U.S.A). Bax antibody, Bcl-2 antibody, CDK1 antibody, CyclinB1 antibody were purchased from Wuhan Sanying Biotechnology Co. P-gp antibody, VEGF antibody, MMP-9 antibody, E-Cadherin antibody were purchased from Wuhan Aipotec Biotechnology Co.

Instruments

CO₂ incubator, benchtop high-speed frozen centrifuge, benchtop centrifuge, enzyme marker were purchased from Thermo Corporation, ultra-clean bench was purchased from Ishiko (Shanghai) Enterprise Development Co.

Cell culture

Cells were cultured in DMEM medium containing 10% FBS, 100 µ/mL penicillin and 0.1 mg/mL streptomycin at 37°C in a 5% CO₂ cell culture incubator. The drug resistance of Siha cells was induced by gradually increasing the concentration of DDP in the culture medium, the concentration of DDP was 0.1µg/mL as the starting point, until the cells could survive for a long time in the medium containing 2 µg/MLDDP.

Drug preparation

(1) Preparation of Anlotinib

50 mg of Anlotinib hydrochloride solid was prepared as a drug stock solution by adding 2.082 mL of DMSO solution, at which time the stock solution was 50 mM. In the experiment, the mother solution was diluted to the required concentration in the corresponding medium; it was ensured that the concentration of DMSO did not exceed 0.1%, and 0.1% DMSO in the medium was used as a control.

(2) Preparation of DDP solution

10 mg of cisplatin was dissolved in 6.25mL PBS(1600 mg/L) and diluted to the required concentration using DMEM medium during the experiment.

Proliferation assay

Siha/DDP cells were divided into control group, experimental group and blank group. The control group contained DMEM medium without anlotinib, the experimental group contained DMEM medium with different concentrations of anlotinib (1.25, 2.5, 5, 10, 20, 40, 80 µM), and the blank group only contained DMEM medium. After 48h of drug treatment, add 10µL of CCK-8 solution to each well and incubated at 37℃ for 1h. The absorbance of the solutions was detected at 450nm.The cell viability rate was calculated as follows: (absorbance of experimental group- absorbance of blank group)/( absorbance of control group- absorbance of blank group) × 100.

Annexin V-FITC/PI double-staining

Siha/DDP cells were inoculated with 5.0×10⁵ cells per well into 6-well plates and cultured for 24h. The cells were divided into control and anlotinib experimental groups (5, 10, 20 µM); after 48h of drug intervention, the cells were harvested by trypsinzation, washed twice with 1×annexin V binding buffer. and then added 5µL each of Annexin V-FITC and PI for 10 min at 25℃ and protected from light. Then added 400µL of 1×binding buffer, mixed well and detected by flow cytometry.

Cell scratching experiment

Siha/DDP cells were inoculated with 5.0×10⁵ cells per well into 6-well plates. After the cell density reached 90%, a straight line was scratched in the center of each well. Washed twice with PBS, then added serum-free medium containing different concentrations of anlotinib to the different groups and continued to incubated for 48h. The moment after scratching was taken as 0h. The cell migration rate was calculated as follows :(1–48 h scratch width/0 h scratch width) × 100.

Cell invasion assay

The matrigel was diluted with DMEM at a ratio of 1∶8, and 50mL diluted solution was dripped in a transwell chamber and placed in an incubator to dry, washed twice with PBS and the number of cells was adjusted to 1.0× 10⁵cells/mL. Added 200µL cells to the upper chamber and 500 µL of medium containing 20% FBS to the lower chamber. After 48h, the gel and cells in the upper chamber were carefully wiped off with a cotton swab and fixed with 4% paraformaldehyde for 30 min, then stained with Crystal Violet solution for 30min, washed twice with PBS, and photographed under a microscope after the chamber was dried, the number of invasive cells was counted from 5 fields randomly.

Western blot analysis

After drug treatment, cells were lysed with cell lysis buffer and the cell lysates were centrifuged at 4°C and 12,000 rpm for 20 min, and then the supernatants were collected. The protein samples were separated by SDS-PAGE(10% or 15%) and transferred onto NC membranes. Subsequently, the NC membranes were blocked with 5%non-fat milk solution and incubated with antibodies overnight at 4℃.After thoroughly washed with 1×TBST, the NC membranes were incubated with respective secondary antibodies. The protein bands were visualized with the ECL prime kit.

Transcriptome sequencing to explore the effect of anlotinib on the gene expression profile of cisplatin-resistant cells in cervical cancer

In the control group, cells were treated with 0.1% DMSO solvent, and in the experimental group, Siha/DDP cells were treated with 10µM anlotinib, with three replicates in each group. After 48h of drug action. Total cellular RNA was extracted with trizol reagent and sequenced by Metware. The RNA-seq data from Siha/DDP cells obtained after sequencing was completed were filtered, and finally the reference genome comparison and expression quantification were performed using Hisat2 and Stringtie software, and the measure of transcript or gene expression level was FPKM.

Differential Gene Screening

For samples with biological replicates, differential expression analysis between sample groups is performed using DESeq2, and for no biological replicates we use edgeR, which requires input of non-normalized reads count data for genes. The reads counts of genes were implemented using featureCounts. After differential analysis, the false discovery rate (FDR) was derived using the Benjamini-hochberg method for multiple hypothesis testing correction of the hypothesis P-value. Differential genes were filtered in the range of |log2Fold Change|≥1 and FDR < 0.05.

Functional Enrichment Analysis of Differential Gene

The GO database was used to analyze the function of differential genes at three levels: biological process, cellular component and molecular function, and then the enrichment pathway of differential genes was analyzed by KEGG. GO analysis and KEGG enrichment analysis of differential genes were also performed with the clusterProfiler package of R software.

Construction of Protein-protein Interaction(PPI) Network

Use stringApp in cytoscape software to export protein interaction networks for differential genes with default values; use MCODE to sort clustering function modules with default values; evaluate networks and calculate scores for each differential gene according to the Degree algorithm of the cytoHubba plugin. Use stringApp in Cytoscape software to export protein interaction networks for differential genes with default parameters; use MCODE to store clustering function modules with default parameters; use cytoHubba's Grau algorithm to evaluate the network and calculate the score for each differential gene.

CCK-8 assay to detect the sensitivity of Siha/DDP cells to DDP

In order to determine the drug resistance of human cervical cancer Siha/DDP cells, Siha and Siha/DDP cells were treated with different concentrations of DDP for 48 h. The results of CCK-8 method showed that the IC50 values of DDP for Siha and Siha/DDP cells were 2.35 µg/mL and 12.73 µg/mL respectively. The resistance index RI = 12.73/2.35 = 5.42, which showed that Siha/DDP showed moderate resistance to cisplatin.

The effect of Anlotinib on the proliferative activity of Siha/DDP cells

When Siha and Siha/DDP cells were treated with different concentrations of anlotinib(0, 1.25, 2.50, 5.00, 10.00, 20.00, 40.00, 80.00 µM) for 48h, the proliferation rates of Siha/DDP cells of action were (103.00 ± 0.33)%, (92.75 ± 2.05)%, (81.75 ± 1.46)%, (69.36 ± 2.32)%, (52.23 ± 1.35)%, (28.11 ± 1.08)%, (13.68 ± 0.88)%, and (5.20 ± 0.56)% respectively. The results showed that anlotinib had an inhibitory effect on the viability of human cervical cancer Siha/DDP cells, and the higher the concentration of anlotinib, the more obvious the inhibitory effect on Siha/DDP cell viability. The IC₅₀ of anlotinib on Siha/DDP cells for 48h was calculated from the proliferation rate data as 9.71 µM.

The effect of anlotinib on apoptosis of Siha/DDP Cells

As shown in Fig. 1a, The total apoptosis rates of Siha/DDP cells were (7.61 ± 1.43)%, (16.63 ± 1.50)%, (33.00 ± 3.39)% and (53.60 ± 3.26)% when the concentration of anlotinib was 0, 5, 10 and 20 µM, respectively. The total apoptosis rate of human cervical cancer Siha/DDP cells increased significantly with the increase of anlotinib concentration, and the difference was statistically significant (P < 0.05). The experimental results indicated that anlotinib had the effect of inducing apoptosis in Siha/DDP cells.

The effect of anlotinib on the migratory capacity of Siha/DDP Cells

Siha/DDP cells were treated with different concentrations of anlotinib and cell scratching experiments were performed. The results are shown in Fig. 1b. When 0, 5, 10 and 20 µM anlotinib were added to intervene Siha/DDP cells, their cell migration rates were (73.80 ± 6.32)%, (52.38 ± 2.40)%, (38.10 ± 3.29)% and (23.79 ± 1.54)% respectively. The ability of anlotinib to inhibit the migration of Siha/DDP cells gradually increased with increasing dose of anlotinib.

The effect of anlotinib on the invasion ability of Siha/DDP cells

As shown in Fig. 1c, the number of cell invasion in 0, 5, 10 and 20 µM anlotinib groups were (192 ± 12), (116 ± 9), (85 ± 5) and (47 ± 4) cells, respectively. The inhibitory effect on Siha/DDP cell invasion was gradually increased with the gradual increase of anlotinib dose.

The effect of anlotinib on Bax, Bcl-2, P21, CDK1 and CyclinB1 protein expression levels in Siha/DDP cells

It was found by western blot that the protein levels of Bax and P21 were significantly increased (P < 0.05) and the protein levels of Bcl-2, CDK1 and CyclinB1 were significantly decreased (P < 0.05) with the increase of the effect concentration of anlotinib compared with the control group(Fig. 1d).

The effect of anlotinib on VEGF, MMP-9, E-cadherin, P-gp protein levels in Siha/DDP cells

Protein immunoblotting data showed that the E-cadherin protein expression level was significantly up-regulated (P < 0.05) with increasing concentration of anlotinib effect compared with the control group, while VEGF, MMP-9 and P-gp protein expression inhibition was also significantly up-regulated (P < 0.05) (Fig. 1e).

Analysis of differentially expressed genes

The differential expression of genes in Siha/DDP cells by anlotinib-treated group and control group, a total of 787 significantly differentially expressed genes were obtained, of which 398 were up-regulated and 389 were down-regulated. Volcano maps and clustering heat maps were constructed for differentially expressed genes. The volcano plot shows the overall gene expression changes in Siha/DDP cell line after treatment with anlotinib, with up-regulated genes in red, down-regulated genes in green and no differentially expressed genes in blue. As seen from the volcano plot, the number of differentially expressed genes up-regulated in Siha/DDP cells under the effect of anlotinib is comparable to the number of down-regulated genes(Fig. 2a). In the clustering heat map, red indicates high expression and green indicates low expression(Fig. 2b).

GO functional enrichment analysis

GO functional enrichment analysis mainly describes the functions of genes in terms of biological process (BP), cellular component (CC) and molecular function (MF). Compared with the control group, the highest GO enrichment in Siha/DDP cells after anlotinib action included regulation of ERK1 and ERK2 cascade, cholesterol biosynthesis process, steroid biosynthesis and metabolic process, extracellular matrix, cellular component of protein-DNA complex, protein heterodimerization molecular function, etc(Fig. 2c).

KEGG pathway enrichment analysis

As seen from the results of KEGG pathway analysis, Siha/DDP cells were significantly enriched by the effects of anlotinib in pathways including neutrophil extracellular traps, alcoholism, systemic lupus erythematosus, steroid biosynthesis, PI3K-Akt signaling pathway and cancer signaling pathway(Fig. 2d).

PPI analysis of differentially expressed genes

The PPI network made by Cytoscape software is shown in Fig. 3a. As shown in Fig. 3b, the 4 key modules screened by MCODE plug-in, the most important module contains 13 node genes and 60 edge numbers, containing genes SQLE, MSMO1, LSS, FDFT1, SC5D, HSD17B7, MVK, HMGCS1, IDI1, FDPS, MVD, NSDHL, HMGCR, among which the genes with degree values greater than 10 were SQLE, MSMO1, LSS, FDFT1 and HMGCS1, and it was speculated that these 5 genes might be the key core genes.

Cervical cancer is one of the most common malignancies affecting women worldwide, seriously affecting their health and quality of life. Anlotinib is a novel oral small molecule multi-target receptor tyrosinase inhibitor developed in China. It can effectively inhibit kinases such as VEGFR, PDGFR, FGFR and stem cell growth factor receptor (c-Kit), with anti-angiogenic and anti-tumor effects(Guanghua and Yu 2022, Lin et al. 2018). The results of this study showed that anlotinib had a significant inhibitory effect on Siha/DDP cell viability, and the higher the concentration, the more pronounced the inhibitory effect on Siha/DDP cell viability. Apoptosis is a self-regulated mode of cell death in living organisms, and its normal functioning affects all life activities in the human body(Chaudhry et al. 2022). The tumorigenic process is a complex combination of changes in normal cell proliferation capacity and apoptotic imbalance. The number of clones formed by Siha/DDP cells gradually decreased and the total apoptosis rate increased significantly with increasing doses of anlotinib. Bcl-2 gene plays an active role in cell death(Qian et al. 2022). Bax is primarily involved in the intrinsic apoptotic signaling pathway and, when activated, aggregates from the cytoplasm to the outer mitochondrial membrane, prompting mitochondrial production of cytochrome C and activating downstream proteins that initiate the apoptotic process(Peng et al. 2020). The pro-apoptotic protein Bax triggers the activation of the intrinsic pathway, which is an excellent target for the development of therapeutic agents that are currently in clinical trials. Cell cycle regulation is achieved by cell cycle proteins in each phase of the cell cycle, among which P21, Cyclin and CDKs are important factors involved in cell cycle regulation. P21 is an oncogene that inhibits cell division and prevents cells from entering S phase from G1 phase through CDK (Meirong and kuanyong 2018, Li-yun et al. 2021). Meanwhile, polymorphisms of P21 gene are associated with tumor susceptibility and can coordinate the relationship between DNA repair, replication and cell cycle(Bingnan et al. 2022). CyclinB1 and CDK1 are core proteins that regulate the G2/M phase of the cell cycle. At the end of S phase, CyclinB1 binds to CDK1 as a regulatory subunit and acts as a mitotic promoter, driving the transition from G2 to M phase(Lizhen 2009). The results of this study showed that the number of cells in the G0/G1 phase and the number of cells in the G2/M phase were significantly reduced and increased after treatment of different doses of anlotinib. The immunoblotting results also showed that the levels of Bax and P21 proteins were significantly increased, and the expression of CDK1, Bcl-2 and CyclinB1 proteins were significantly decreased after the effect of anlotinib. These results indicated that anlotinib could inhibit the proliferation and promote the apoptosis of Siha/DDP cells.

In patients with cervical cancer, treatment failure is also attributed to a variety of ways in which tumor cells can metastasize and spread to adjacent and distant organs, causing cancerous lesions. In this study, we found that the inhibition of cell migration and invasion increased with increasing doses of anlotinib. Epithelial-mesenchymal transformation (EMT) is the loss of epithelial properties of tumor cells and the acquisition of mesenchymal properties, mainly in the form of reduced expression of the epithelial marker E-cadherin, which is closely associated with tumor invasion and metastasis and plays an important role in resistance to many antitumor drugs(Jing and Mao 2022). The growth and metastasis of malignant tumors depend on the formation of new blood vessels. VEGF is a class of cell-secreted growth factors that stimulates the formation of blood vessels and lymphatic microtubules, and plays an important role when tissue oxygenation is insufficient(Chuai et al. 2021). Matrix metalloproteinase is an endopeptidase activity that degrades proteins in the extracellular matrix, and its expression affects tumor development(Sidorkiewicz et al. 2019). MMP-9 can damage collagen in the basement membrane of the blood vessels surrounding type IV tumor cells to spread to surrounding tissues and promote metastasis(Kang et al. 2022). VEGF and MMP-9 are expressed at high levels in cervical cancer, and the higher the serum VEGF and MMP-9 levels, the worse the prognosis of patients. The results of this study showed that anlotinib intervention significantly upregulated E-cadherin protein expression and inhibited VEGF and MMP-9 protein expression. This indicates that anlotinib can inhibit the invasion and migration of Siha/DDP cells. Cervical cancer cells can develop resistance to cisplatin in several ways, including reducing cisplatin uptake and increasing efflux, counteracting the effects of cisplatin on DNA and inhibiting apoptotic signaling pathways. P-gp is an adenosine triphosphate-dependent transmembrane transport protein, and the higher the amount of P-gp in the body, the more likely it is that cancer cells will develop resistance to the drug(Xin-xin et al. 2021, Zhang et al. 2012). In this study, we found that the P-gp protein content in Siha/DDP cells was significantly reduced after treatment with anlotinib, which showed that anlotinib could effectively reduce the drug resistance ability of Siha/DDP.

The research and development of anticancer drugs has traditionally been a major issue in the medical field, due to the fact that the understanding of the mechanisms of cancer development is still very limited(Chao et al. 2020). Through our study, we found that the proliferation, invasion, and migration abilities of Siha/DDP cells were significantly reduced after treatment by anlotinib. To initially explore the mechanisms causing this altered biological function of cervical cancer cells, we sequenced the transcriptome and performed bioinformatic analysis of RNA-seq data. The results showed that the gene expression of Siha/DDP cells was altered after anlotinib intervention, and a total of 787 differential genes were obtained, of which 398 were up-regulated and 389 were down-regulated, suggesting that anlotinib may mediate the biological function of cervical cancer cells by regulating these identified differential genes, thereby inhibiting the development of cervical cancer. Based on the above studies, we further investigated the biological processes and related pathways associated with these differential genes. The GO analysis showed that these genes were mainly enriched in the regulation of ERK1 and ERK2 cascade, collagen-containing extracellular matrix, DNA packaging complex nucleosomes, protein-DNA complex cellular composition, protein heterodimerization molecular function, cholesterol biosynthesis process, ERK1 and ERK2 cascade reaction and steroid biosynthesis and metabolism process, etc. The above results suggest that anlotinib may be involved in different biological processes by regulating different differential genes and performing multiple molecular functions to inhibit the malignant development of cervical cancer. KEGG enrichment analysis revealed that these differential genes were mainly enriched in neutrophil extracellular traps, alcoholism, systemic lupus erythematosus, steroid biosynthesis, PI3K-Akt signaling pathway and cancer-related signaling pathways. Anlotinib may activate or inhibit the above biological pathways by regulating different differential genes, thus inhibiting the abnormal biological activity of cervical cancer cells. Four key modules were screened using the MCODE plug-in in cytoscape, and five key core genes, namely SQLE, MSMO1, LSS, FDFT1 and HMGCS1, were screened. Multiple studies have shown that the functions of these five genes are closely related to the development of cervical cancer.

In summary, anlotinib inhibited the proliferation, migration, and invasion of Siha/DDP cells and promoted their apoptosis. After treatment with anlotinib, Siha/DDP cells caused changes in gene expression profile, resulting in differential expression of several genes including SQLE, MSMO1, LSS, FDFT1 and HMGCS1. It provides a new theoretical basis for the clinical treatment of cervical cancer with anlotinib alone and its combination with cisplatin. However, this study only screened the key genes of anlotinib in cervical cancer resistant cells, but did not validate them yet. The next step is to explore the regulatory role of these key genes in the related pathways through gene knockdown, gene overexpression and the use of pathway inhibitors.

Acknowledgment and funding

We would like to give our sincere appreciation to the reviewers for their helpful comments on this article. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Author contribution

N.N.X. designed studies, analyzed, and wrote the manuscript. M.M.Y. revised and took charge of supervising the manuscript. S.Y.C. and Y.L. collated the data and contributed to drafting of the manuscript. All authors have read and approved the final submitted manuscript.

Data availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

The authors declare that they have no conflict of interest.

Consent for Publication

All authors have read and agreed to the published version of the manuscript.

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Effect and Mechanism of Anlotinib on the Malignant Biological Behavior of Cisplatin-resistant Cells in Cervical Cancer

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Abstract

Purpose

Materials and Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Cell line

The main reagents

Instruments

Cell culture

Drug preparation

Proliferation assay

Annexin V-FITC/PI double-staining

Cell scratching experiment

Cell invasion assay

Western blot analysis

Differential Gene Screening

Functional Enrichment Analysis of Differential Gene

Construction of Protein-protein Interaction(PPI) Network

Results

CCK-8 assay to detect the sensitivity of Siha/DDP cells to DDP

The effect of Anlotinib on the proliferative activity of Siha/DDP cells

The effect of anlotinib on apoptosis of Siha/DDP Cells

The effect of anlotinib on the migratory capacity of Siha/DDP Cells

The effect of anlotinib on the invasion ability of Siha/DDP cells

The effect of anlotinib on VEGF, MMP-9, E-cadherin, P-gp protein levels in Siha/DDP cells

Analysis of differentially expressed genes

GO functional enrichment analysis

KEGG pathway enrichment analysis

PPI analysis of differentially expressed genes

Discussion

Conclussion

Declarations

References

Additional Declarations

Status:

Version 1